This project will use single channel patch clamp methods to examine the basic characteristics and the regulation of Na + and Cl- channels in epithelial tissue from normal and CF cells. The preparations which will be used are primary cultures derived from bronchial scrapings or nasal polyps and anepithelial call line ch expresses the CFTR protein (A6, originally derived from the distal nephron of Xenopus laevis). The rationale for the project is based on the observation that there are similar regulatory pathways for both amiloride-blockable sodium channels and outwardly-rectifying chloride channels in A6 cells. These results may have specific relevance to CF since the CF defect manifests itself as an abnormality in the regulation of both sodium and chloride channels. Therefore, the immediate goal of this application is to continue an examination of ion channel regulation using single channel methods in A6 cells and, in addition, extend our studies to an examination of similar channels in airway epithelial cells from cystic fibrosis patients. The specific aims of the proposal are (1) further examine the mechanisms for both sodium and chloride channel regulation in epithelial cells (including airway cells) placing particular emphasis on regulation which involves membrane-associated proteins and their metabolic products including guanine nucleotide-binding proteins and membrane-associated lipases and their metabolites (phospholipase A2 and arachidonic acid; phospholipase C and diacylglycerol); (2) regulation which involves phosphorylation/dephosphorylation by determining the effects of phorbol esters, diacyl glycerols, catalytic subunits of protein kinases, phosphatases and inhibitors of these agents. The important issue will be whether phosphorylation/dephosphorylation modulates any of the membrane-associated proteins associated with the CF defect which produce abnormal regulation of chloride and sodium channels; and (3) confirm the role of specific regulatory mechanisms in CF airway epithelial cells.